Bacteria have evolved to remain attached to infection sites even in the presence of strong mechanical perturbations, such as those induced by mucus flow and coughing in the mucosa, or chewing and brushing in the mouth. Although several adhesive structures have been described in bacteria, very little is known about the molecular mechanisms responsible for the high mechanical endurance of bacteria-host adhesion sites. The main reason is the absence of classical bulk experiments that can probe adhesive junctions under force, greatly limiting our understanding of junctions in vivo and, more importantly, preventing us from developing drugs that target adhesion of pathogenic bacteria. Here, we propose to develop novel single-molecule techniques based on robust mechanical fingerprints that will unambiguously probe the behavior of adhesive junctions that lead to infection, all under physiologically relevant mechanical perturbations. We will consider various types of adhesive interactions involving the pili (fimbriae) of three gram-positive organisms: Corynebacterium diphtheriae (diphtheria), Streptococcus agalactiae (pre-natal infections), and Actinomyces oris (dental plaques). Gram positive bacteria are unique because their pili are assembled as a single continuous polypeptide of repeating folded units that can grow up to several micrometers in length. It is unknown how a single tandem modular protein of that size can withstand large mechanical forces. The proposed new single-molecule assays are based on recent milestone technologies that allow reliable mechanical tethering of proteins to surfaces and on our extensive experience studying the mechanics of proteins using force-spectroscopy instrumentation, both with AFM and magnetic tweezers. Our aim is to identify the Achilles heels of adhesive junctions, i.e. those molecular elements that are essential to the endurance of the junction in vivo. We will measure their mechanical properties and how they mature into fully functional elements in bacterial pili. For instance, we will use our recently developed singl-molecule oxidative folding and mechanical memory assays to examine how mechanically stable disulfide bonds are introduced and modified in pilins of Gram-positive bacteria. We will also combine our HaloTag covalent anchor with Magnetic tweezers to make daylong recordings of the mechanics of intact pili in living bacteria. Our findings will be used to construct a computational model for gram-positive pili that incorporates all of the Achilles heels identified in this proposal and combines them with the physics of an extending polypeptide. We will use Brownian Dynamics applied to our model to predict the mechanical behavior of pili in response to physiological shocks such as coughing. Our model will serve as a quantitative platform for the identification of a novel class of antibiotics and vaccines that work by blocking the ability of bacteria to adhere to their target tissues. Given the rapid growth of bacteria that are resistant t the current classes of antibiotics, developing novel approaches for blocking bacterial infections is an urgent endeavor of great importance to society.